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A tumor marker detection kit based on force coding and its application

A kit and coding sequence technology, applied in the field of biochemical analysis, can solve problems such as difficulty in meeting actual detection requirements, low sensitivity, and high technical requirements

Active Publication Date: 2020-12-15
INST OF CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the methods based on nucleic acid aptamers to detect MUC1 and HER2 mainly include fluorescence resonance energy transfer, electrochemical method, surface plasmon resonance technology and absorbance method, etc., but these methods have high technical requirements and low sensitivity, and it is difficult to meet the actual detection requirements.
Moreover, based on the detection of these methods, the range of detection objects cannot be well regulated.
And when multiple proteins are detected at the same time, due to the large difference in the content of these proteins in the body (from pM level to uM level), the practical application of these methods is limited.

Method used

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  • A tumor marker detection kit based on force coding and its application
  • A tumor marker detection kit based on force coding and its application
  • A tumor marker detection kit based on force coding and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Embodiment 1: Preparation of aldehydized glass substrate

[0115] The cut glass slices were ultrasonically washed twice with acetone, then ultrasonically washed three times with ultrapure water, ultrasonically washed with 1mol / L NaOH solution for 10min, and then washed twice with ultrapure water and absolute ethanol respectively. Place it in a drying oven at 250°C for 3 hours to remove bacteria on the surface.

[0116] Add the sterilized glass substrate into the newly prepared Piranha solution beaker (98% concentrated H 2 SO 4 : 30%H 2 o 2 =7:3, the volume ratio of the two), put the above-mentioned beaker in a 90°C constant temperature water bath and let it soak for 30min, and the surface of the glass sheet is hydroxylated.

[0117] The glass substrate just hydroxylated above was added into a toluene solution containing 1% APTES, soaked at room temperature for 3 hours, and the glass substrate was aminated. Wash the surface with toluene solution for 3 times, then wa...

Embodiment 2

[0119] The fixation of embodiment 2 nanometer sensor

[0120] Add 20 μL of 3 μM aminated MUC1 force coding sequence and 3 μM aminated HER2 force coding sequence at a volume ratio of 1:1 to the surface of the aldylated glass substrate prepared in Example 1, reduce with sodium borohydride, and incubate overnight at 4°C.

[0121] Mix equal volumes of 3 μM MUC1 aptamer and 3 μM complementary DNA sequence (Com 13), react in a metal bath at 90°C for 5 min, and then allow it to cool down to room temperature naturally to obtain the hybridized MUC1 first hybrid nucleic acid (hereinafter Abbreviated as DNA1). Mix equal volumes of 3 μM HER2 nucleic acid aptamer and 3 μM complementary DNA sequence (Com 16), react in a metal bath at 90°C for 5 min, and then allow it to cool down to room temperature naturally to obtain the hybridized HER2 first hybrid nucleic acid (following Abbreviated as DNA2).

[0122] Take 20 μL of DNA1 and DNA2 with a volume ratio of 1:1 and add them to the sample po...

Embodiment 3

[0123] Embodiment 3 force spectrum measurement

[0124] This embodiment provides a method for force spectrum measurement:

[0125] (1) Fixed MUC1 nanosensor:

[0126] The specific steps are the same as those in Example 2, except that the HER2 nanosensor is not included, and only the MUC1 sensor is fixed on the substrate.

[0127] (2) Measure the force spectrum of MUC1:

[0128] The glass substrate fixed with the nanosensor obtained in step (1) was centrifuged at 1500 rpm / min for 5 minutes (the direction of the centrifugal force suffered by the nanosensor is directed from the substrate to the nanosensor) to remove physical adsorption. The magnetic signal is then detected on an ultra-low field atomic magnetometer. Gradually increase the magnitude of the centrifugal force, and the centrifugation time is still 5 minutes each time, and record the magnetic signal intensity corresponding to each centrifugal force. Due to the non-covalent bond between double-stranded DNA, under th...

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Abstract

The invention relates to a tumor marker detection kit based on force coding and application of the tumor marker detection kit. The tumor marker detection kit comprises one or more nano sensors, magnetic beads and a solid phase substrate coupled with or fixed to the nano sensors; and each nano sensor comprises a nucleic acid aptamer, a force coding sequence and a complementary DNA sequence. The tumor marker detection kit based on force coding detects a target by detecting magnetic signals, compared with traditional fluorescence signals, interference of ambient light and the solution color is avoided, and detection is sensitive; and the kit can simultaneously detect a plurality of unmarked tumor markers, and the problems that different proteins have the large concentration difference in vivoand cannot be clinically detected are overcome. In addition, the kit can further detect small molecules, nucleic acid and the like besides detecting the proteins and has wide application prospects.

Description

technical field [0001] The invention relates to the technical field of biochemical analysis, in particular to a tumor marker detection kit based on force coding and its application. Background technique [0002] Cancer is a disease that seriously threatens human health. Millions of people die from cancer every year in the world, and its incidence is increasing year by year, especially the incidence of lung cancer, breast cancer, gastric cancer, liver cancer and colorectal cancer tops the list. According to statistics, at present, among all new cancer patients every year, the incidence of female breast cancer has accounted for a quarter of all tumor diseases, and the mortality rate caused by malignant cancer is also the highest. [0003] MUC1 is a member of the mucin family, and its coding gene is located at the 1q21 locus of human chromosome. MUC1 is a multifunctional macromolecular glycoprotein with a complex glycosyl structure. It is mainly distributed on the surface of v...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6825C12Q1/6834
CPCC12Q1/6825C12Q1/6834C12Q2525/205C12Q2563/143C12Q2563/149C12Q2565/607
Inventor 姚立鲁攀于婵婵
Owner INST OF CHEM CHINESE ACAD OF SCI
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